Jiangtao HAO, Yuan JING, Chaochan LI

Guizhou Provincial Key Laboratory of Mountainous Environmental Protection, Guizhou Normal University, Guiyang 550001, China

Abstract [Objectives] The spatial distribution characteristics of organic acids in two late-blooming Rhododendron species (Rhododendron decorum and Rhododendron stamineum) in Guizhou Baili Rhododendron National Forest Park were explored, in order to provide reference for exploring the plant-soil relationship of subtropical forest. [Methods] The fresh leaf, stem, root, litter, humus and soil samples of R. decorum and R. stamineum were collected. The contents of eight low molecular weight organic acids including oxalic acid, tartaric acid, malic acid, citric acid, acetic acid, lactic acid, succinic acid and formic acid were determined by high performance liquid chromatography (HPLC). [Results] Oxalic acid is the main organic acid in the two species of Rhododendron. Among different samples, the content of organic acids was in the following order: root > fresh leaf > humus > litter > stem > soil. [Conclusions] The content of organic acids in the root was significantly higher than that in other parts. The types of organic acids in stems were the least.

Key words Subtropical forest, Low molecular weight organic acids, Spatial distribution

1 Introduction

Low molecular weight organic acids (LMWOAs) are ubiquitous in forest environment and plant organs. They are important intermediates in the metabolic pathway of organic matter and play an important role in the plant-soil material cycle[1-2]. LMWOAs are also common in forests. They are continuously released into the soil from root exudates, microbial activities and decomposition of organic matter. The concentration of LMWOAs in soil was usually in the range of 10-6-10-3mol/L[3]. Although it is not high, as regulating substances, LMWOAs have a profound impact on the chemical and biological processes of forest soil and plant growth by dissolving and converting insoluble nutrients through chelation and ligand exchange[4-6]. At present, the research on forest LMWOAs mostly focuses on root exudates and litter, and more progress has been made in the role of LMWOAs in forest soil physical, chemical and biological processes[7-8]. However, there is little literature on the spatial distribution of LMWOAs in forest plants.

RhododendrondecorumFranch (RD) andRhododendronstamineumFranch (RS) in Guizhou BailiRhododendronNational Forest Park (hereinafter referred to as "BailiRhododendron") bloom from May to June every year, and belong to late-blooming species, and they prolong the viewing time in the park and have important ornamental and economic value[9-12]. At present, scholars have reported the progress of cutting propagation, rapid propagation and metabolites of the twoRhododendronspecies[13-15], but there is no report on LMWOAs of late-bloomingRhododendronspp. In this paper, theR.decorumandR.stamineumin BailiRhododendronare taken as the research objects. The contents of LMWOAs inRhododendronroots, stems, and leaves, litter, humus and soil were determined to explore the spatial distribution characteristics of LMWOAs in late-bloomingRhododendronspecies, so as to provide scientific basis for more reasonable protection and development of late-bloomingRhododendronresources.

2 Materials and methods

2.1 Study siteThe test site is located in Baili Rhododendron (105°50′16″-106°04′57″ E, 27°10′07″-27°17′55″ N, 1 060-2 200 m a.s.l.). There are more than 40 kinds of rhododendrons (including subspecies and varieties) in the park, with the largest primary rhododendron forest identified in China and even the world[16]. Baili Rhododendron is located in the northwest of Guizhou Province. It is located in the slope zone of the transition from the western Guizhou Plateau to the middle mountains of Guizhou. The climate belongs to the middle subtropical warm and humid monsoon climate, with an average annual temperature of 11.8 ℃ and an average annual relative humidity of 84%. The annual precipitation is 1 000-1 100 mm, the rainy season is from May to October, and the dry season is from November to April of the next year. The annual average sunshine hours are 1 335.5 h[5].

2.2 Research methods

2.2.1Collection of samples and determination of soil physical and chemical indexes. Taking theR.decorumandR.stamineumstand in Baili Rhododendron as the research object, three sample plots with a size of 5 m × 5 m were randomly selected and the basic situation of the sample plots was investigated (Table 1). The fresh leaves, stems, roots, litter, humus and soil (0-10 cm) samples were collected. Then, the samples of each plot were mixed, put into plastic bags, stored in ice boxes and taken back to the laboratory in success. After the samples were naturally dried, they were ground and passed through a 100-mesh sieve. The soil temperature was measured by portable soil thermometer, the relative water content of soil was measured by drying method (105 ℃), and the pH of soil was measured by Potentiometric method (water-soil ratio 2.5∶1)[17].

Table1 Basic situation of different rhododendron forest sample plots

2.2.2Determination of LMWOAs. A certain amount (1 g) of each sample was poured into a 20 mL conical flask, added 5 mL of 0.1% phosphoric acid solution , placed in a constant temperature culture oscillator for 24 h, and let stand for 5 min in success. Subsequently, the supernatant was collected and centrifuged at 6 000 r/min for 10 min. The new supernatant was passed through 0.22 μm aqueous microporous filter membrane, and the content of LMWOAs in the filtrate was determined by high performance liquid chromatography (HPLC). The detection conditions refer to the method in document[7].

2.2.3Instruments and reagents. The main instruments and drugs used in the experiment are shown in Table 2.

Table 2 Details of instruments and reagents

2.3 Data processingData analysis was performed using SPSS 19.0, Microsoft Excel 2016, and Origin 2019 software.

3 Results and analysis

3.1 Distribution characteristics of organic acids in two late-bloomingRhododendronspeciesAs shown in Table 3, the contents of oxalic acid, tartaric acid, malic acid, lactic acid and organic acids in roots, stems and leaves of the twoRhododendronspecies were significantly different (P<0.05), and the contents of oxalic acid, tartaric acid and organic acids all ranked as roots>fresh leaves > stems. The content of organic acids in the roots of rhododendron pistil was the highest, 748.11 μg/g, and the content of organic acid in the stems of rhododendron grandiflorum was the lowest, which was 313.78 μg/g. Oxalic acid was the most low molecular weight organic acid and succinic acid was the least.

Table 3 Distribution characteristics of LMWOAs in late-blooming Rhododendron species

3.2 Distribution characteristics of organic acids in three soil layersThe distribution of LMWOAs in late-bloomingRhododendronwas determined as shown in Table 4. The contents of oxalic acid, formic acid, acetic acid, citric acid and organic acids inR.decorumwere in the order as humus > litter > soil, while the contents of oxalic acid, tartaric acid, malic acid, citric acid, acetic acid, lactic acid, succinic acid, formic acid and organic acids inR.stamineumranked as litter > humus > soil, and the difference was significant (P<0.05). The contents of various LMWOAs in the litter and humus of the two late-bloomingRhododendronspecies were significantly higher than those in the soil (P<0.05).

Table 4 Distribution characteristics of LMWOAs in three soil layers

3.3 Differences and characteristics of LMWOAs in two late-bloomingRhododendronspeciesThe types and contents of LMWOAs in each part of the two late-bloomingRhododendronforests were quite different (Fig.1). The eight kinds of LMWOAs in roots, fresh leaves, litter, humus and soil of the twoRhododendronforests were detected, and the types of organic acids in stems were the least. Oxalic acid was the main organic acid in two kinds of late-bloomingrhododendron, and its average relative content was 40.6%. The proportion of oxalic acid inR.decorumwas as high as 57%; the proportions of tartaric acid and malic acid were the second; and the content of succinic acid in each part was low, and its relative content was no more than 5%. The proportion in the roots ofRhododendronwas the highest, which was 3.6%.

Fig.1 Distribution and composition of LMWOAs in two late-blooming Rhododendron species

The spatial distribution of organic acids in late-bloomingRhododendronwas roots > fresh leaves > humus > litter > stems > soil (Fig.2). Except that there was no significant difference in total organic acid content between litter and humus, soil and stem, there was a significant difference in total organic acid content between other parts (P<0.05). The total content of organic acids in roots was the highest, which was 748.11 μg/g, and that in soil was the lowest, which was 304.76 μg/g.

Note: Different lowercase letters on the top of the columns indicate significant differences at the 0.05 level.

4 Discussion

LMWOAs are widely distributed in the forest environment, and their species and content are affected by plant species, natural environment, human interference and other factors[18-19]. Researchers have detected a variety of LMWOAs such as oxalic acid, citric acid, lactic acid, formic acid and malonic acid in the roots, stems, leaves, litter, humus and soil of forest plants[18-20]. In this study, oxalic acid, tartaric acid, formic acid, malic acid, lactic acid, acetic acid, citric acid and succinic acid were detected in fresh leaves, roots, litter and soil of two late-bloomingRhododendronforests[21-22]. However, there are few kinds of organic acids detected in plant stems, and the content of oxalic acid is the least in plants, which is consistent with the results of previous research[23]. In different parts of the twoRhododendronspecies, it is found that oxalic acid has the highest content and is the main LMWOAs, which is consistent with the previous research results[24].

The sources of LMWOAs in forest are complex and they are continuously released into soil through root exudates, microbial metabolites and organic matter decomposition[25]. The concentration of LMWOAs in soil depends on the balance between entering the soil and being degraded. Most LMWOAs are rapidly degraded by microorganisms in soil[11]. In this study, it was found that the spatial distribution of LMWOAs in differentRhododendronforests was: roots > fresh leaves > humus > litter > stems > soil. The content of LMWOAs in roots is the highest and the content in soil is the lowest, consistent with the previous research results[23]. Studies have shown that low phosphorus stress promotes the increase of LMWOAs secreted by plant roots, especially oxalic acid, citric acid and malic acid[26]. Therefore, the high content of oxalic acid and malic acid in this study may be due to the low content of phosphorus in soil environment.

LMWOAs can reduce the content of nutrients and base ions in soil through the activation of soil nutrient elements, thus aggravating the acidification of acidic soil[27]. Recent studies found that oxalic acid, citric acid and tartaric acid are easy to accelerate soil acidification[28]. In this study, the soil pH values of the two late-bloomingRhododendronforests were 4.75 and 3.86, respectively, which may be affected by the concentration of LMWOAs in soil and roots.

5 Conclusions

In this paper, the contents of organic acids in two kinds of late-blooming rhododendron were determined. There were obvious differences in the spatial distribution of organic acids. Among different samples, the total organic acid content ranked as roots > fresh leaves > humus > litter > stems > soil. The content of organic acids in roots was significantly higher than that in other parts. Oxalic acid was the main LMWOAs in the two late-bloomingRhododendronforests.